EP2852415A1 - Lipid nanoparticle compositions for antisense oligonucleotides delivery - Google Patents

Lipid nanoparticle compositions for antisense oligonucleotides delivery

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Publication number
EP2852415A1
EP2852415A1 EP13726404.0A EP13726404A EP2852415A1 EP 2852415 A1 EP2852415 A1 EP 2852415A1 EP 13726404 A EP13726404 A EP 13726404A EP 2852415 A1 EP2852415 A1 EP 2852415A1
Authority
EP
European Patent Office
Prior art keywords
lipid nanoparticle
cancer
nanoparticle composition
nucleic acid
hif
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13726404.0A
Other languages
German (de)
English (en)
French (fr)
Inventor
Robert J. Lee
Young Bok Lee
Deog Joong Kim
Chang Ho Ahn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ohio State University
Original Assignee
Ohio State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ohio State University filed Critical Ohio State University
Publication of EP2852415A1 publication Critical patent/EP2852415A1/en
Withdrawn legal-status Critical Current

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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61K38/08—Peptides having 5 to 11 amino acids
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
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    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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Definitions

  • the present disclosure describes lipid nanoparticles usable for the delivery of nucleic acids and related compounds.
  • siRNA and other therapeutic oligonucleotides are a major technical challenge that has limited their potential for clinical translation.
  • a liposome is a vesicle composed of one or more lipid bilayers, capable of carrying hydrophilic molecules within an aqueous core or hydrophobic molecules within its lipid bilayer(s).
  • Lipid nanoparticles is a general term to described lipid-based particles in the submicron range. They can have structural characteristics of liposomes and/or have alternative non-bilayer types of structures. Drug delivery by LNs via systemic route requires overcoming several physiological barriers. The reticuloendothelial system (RES) can be responsible for clearance of LNs from the circulation. Once escaping the vasculature and reaching the target cell, LNs are typically taken up by endocytosis and must release the drug into the cytoplasm prior to degradation within acidic endosome conditions.
  • RES reticuloendothelial system
  • LNs with a highly positive charge tend to interact non-specifically with target cells and circulating plasma proteins, and may cause cytotoxicity.
  • LNs with a highly negative charge cannot effectively incorporate nucleic acids, which are also negatively charged, and may trigger rapid RES-mediated clearance, reducing therapeutic efficacy.
  • LNs with a neutral to moderate charge are best suited for in vivo drug and gene delivery.
  • LNs constitute a promising platform for the delivery of traditional therapeutic compounds and nucleic acid-based therapies.
  • Drugs formulated using LNs can often feature superior pharmacokinetic (PK) properties in vivo, such as increased blood circulation time and increased accumulation at the site of solid tumors due to enhanced permeability and retention (EPR) effect.
  • PK pharmacokinetic
  • LNs may be surface-coated with polyethylene glycol to reduce opsonization of LNs by serum proteins and the resulting RES-mediated uptake.
  • LNs can also be coated with cell-specific ligands to provide targeted drug delivery.
  • Nucleic acid-based therapies work on the premise of introducing nucleic acids (NAs) to promote or inhibit gene expression. As mutations in genes and changes in miRNA profile are believed to be the underlying cause of cancer and other diseases, nucleic acid-based agents potentially can directly act upon the underlying etiology, maximizing therapeutic potential.
  • a few examples of nucleic acid- based therapies include plasmid DNA (pDNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miR) mimic (or mimetic), anti-miR/antagomiR/miR.
  • nucleic acid as used in the present disclosure.
  • the clinical translation of nucleic acid-based therapies faces several obstacles in its implementation. Transporting nucleic acids to their intracellular target is particularly challenging as nucleic acids are relatively unstable and are subject to degradation by serum and cellular nucleases. Further, the high negative charges of nucleic acids make it impossible for transport across the cell membrane, limiting utility. Viral vectors have been developed to address this issue, but most have failed due to activation of immunological responses in vivo and induction of undesired mutations in the host genome. Non-viral vectors have also been investigated extensively, but few have yielded successful clinical outcomes and further improvements are needed.
  • cationic LNs have been utilized as non-viral vectors for gene delivery.
  • cationic lipids are replaced with, or used in combination with, anionic lipids.
  • the positive charge of cationic LNs facilitates an electrostatic interaction with negatively charged nucleic acids.
  • Anionic lipids can be combined with cationic lipids or with a cationic polymer, which will in turn mediate interaction with the nucleic acids. These may be prepared by various techniques known in the art such as ethanol dilution, freeze-thaw, diafiltration, and thin film hydration.
  • LNs are typically composed of helper lipids, including bilayer- forming phospholipid components such as phosphatidylcholines, as well as cholesterol.
  • Helper lipids such as dioleoylphosphatidylethanolamines (DOPE) do not favor bilayer phase and instead aid in disrupting the lipid bilayer at the target site to release the therapeutic agent.
  • Stabilizing components such as D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS), which is a PEGylating agent, or mPEG-DSPE may be added to stabilize the formulation and protect the LN from RES-mediated uptake.
  • TPGS D-alpha tocopheryl polyethylene glycol 1000 succinate
  • mPEG-DSPE may be added to stabilize the formulation and protect the LN from RES-mediated uptake.
  • lipid nanoparticle formulation should be able to (1) protect the drug from enzymatic degradation; (2) traverse the capillary endothelium; (3)
  • lipid nanoparticles that can encapsulate therapeutic oligonucleotides with high efficiency and fulfill physical and biological criteria for efficacious delivery.
  • certain embodiments includes lipid nanoparticles containing RX- 0201(Archexin ® ), which is a 20-mer phosphorothioate antisense oligonucleotide having a sequence that includes 5' gctgcatgatctccttggcg 3' (Seq. Id. No.: 1) against Akt-1, and/or RX-0047, which is a 20-mer phosphorothioate antisense oligonucleotide having a sequence that includes
  • the lipid nanoparticles comprise hyper-cationized and/or pH- responsive HSA-polymer conjugates.
  • the HSA-polymer conjugate comprises HSA-PEHA.
  • the lipid nanoparticles hyper-cationized albumin- polymer conjugates (APC) in order to increase the transfection efficiency of lipid nanoparticle formulations.
  • compositions methods of making a lipid-coated albumin nanoparticles, and methods of treating a cancer or other disease.
  • the present invention is a lipid nanoparticle composition that includes a macromolecule conjugated to a polymer and a targeting agent.
  • the lipid nanoparticle composition also includes a therapeutic agent such as nucleic acids, proteins, polysaccharides, lipids, radioactive substances, therapeutic agents, prodrugs, and combinations thereof.
  • the therapeutic agent is a nucleic acid.
  • the nucleic acid is pDNAs, antisense oligonucleotides, miRs, antimiRs, shRNAs, siRNAs, or combinations thereof.
  • the therapeutic agent is an antisense oligonucleotide (ASO) that can be an ASO targeted to a portion of a nucleic acid encoding Akt-1, and which modulates the expression of Akt-1 ; or an ASO targeted to a portion of a nucleic acid encoding HIF-1, and which modulates the expression of HIF- 1.
  • ASO antisense oligonucleotide
  • Embodiments of the present invention also include a lipid nanoparticle composition that includes a macromolecule conjugated to a polymer and a therapeutic agent that is an ASO such as an ASO targeted to a portion of a nucleic acid encoding Akt-1, and which modulates the expression of Akt-1 or an ASO targeted to a portion of a nucleic acid encoding HIF-1, and which modulates the expression of HIF- 1.
  • a lipid nanoparticle composition that includes a macromolecule conjugated to a polymer and a therapeutic agent that is an ASO such as an ASO targeted to a portion of a nucleic acid encoding Akt-1, and which modulates the expression of Akt-1 or an ASO targeted to a portion of a nucleic acid encoding HIF-1, and which modulates the expression of HIF- 1.
  • the polymer can be charged, for example the polymer can be positively charged.
  • the macromolecule includes albumin.
  • the macromolecule conjugated to a polymer is an albumin-polycation conjugate. Conjugation can be, for example, via cross linking agents.
  • the macromolecule or positively charged polymer can be, for example, pentaethylenehexamine (PEHA), tetraethylenehexamine, and tetraethylenepentamine (TEPA).
  • the macromolecule includes pentaethylenehexamine (PEHA) and the polymer includes human serum albumin (HSA).
  • the ratio of PEHA molecules to HSA molecules can be about 1 1 to 1.
  • the lipid nanoparticle of the invention can include a mixture of two or more low molecular weight polymers.
  • nanoparticle can include DOTAP, SPC, and TPGS, for example a molar ratio of
  • DOTAP:SPC:TPGS at about 25:70:5.
  • the antisense oligonucleotide is a compound having a sequence that includes 5' gctgcatgatctccttggcg 3'(Seq. Id. No.: 1), targeted to a nucleic acid molecule encoding human Akt-1, and which modulates the expression of Akt-1.
  • the antisense oligonucleotide is a compound having a sequence that includes 5' gctgcatgatctccttggcg 3'(Seq. Id. No.: 1), targeted to a nucleic acid molecule encoding human Akt-1, and which modulates the expression of Akt-1.
  • the antisense oligonucleotide is a compound having a sequence that includes
  • the lipid nanoparticle composition can also include a fusogenic peptide.
  • the lipid nanoparticle has a particle size under about 300 nm or under about 150 nm.
  • the targeting agent can be bound only to an external surface of the lipid nanoparticle via direct connection or via a crosslinker.
  • the targeting agent can be an antibody or an antibody fragment.
  • the targeting agent can also be a cRGD peptides, galactose-containing moieties, transferrin, folate, low density lipoprotein, and epidermal growth factors.
  • the targeting agent is cRGDfC, or folate.
  • the targeting agent can be a conjugate such as folate-PEG-CHEMS (folate- polyethylene glycol-cholesteryl hemisuccinate), folate-PEG-DSPE (folate-polyethylene glycol-distearoyl phosphatidylethanolamine), or cRGDfC-PEG-DSPE
  • the invention is a pharmaceutical composition comprising a lipid nanoparticle composition as described above and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition can be prepared as a sterile solution or suspension.
  • the invention is a method of making a lipid-coated albumin nanoparticle (LCAN), wherein the method includes the steps of synthesizing a HSA-PEHA conjugate; preparing a mixture of lipids; adding the mixture of lipids to the HSA-PEHA conjugate; and adding an antisense oligonucleotide (ASO) to the mixture of lipids and the HSA-PEHA conjugate to obtain an LCAN precursor; wherein the ASO is selected from the group consisting of an ASO targeted to a portion of a nucleic acid encoding Akt-1 , and which modulates the expression of Akt- 1 ; and an ASO targeted to a portion of a nucleic acid encoding HIF- 1 , and which modulates the expression of HIF- 1.
  • ASO antisense oligonucleotide
  • the invention is a method of making a lipid-coated albumin nanoparticle (LCAN), that includes the steps of synthesizing a HSA-PEHA conjugate; preparing a mixture of lipids; and adding a targeting agent and the mixture of lipids to the HSA- PEHA conjugate.
  • the targeting agent can be any of the targeting agents as described above.
  • mixture of lipids comprises and targeting agent includes DOTAP, soyPC, TPGS, and cRGDfC-PEG-DSPE, for example wherein the molar ratio of DOTAP:soyPC:TPGS:cRGD-PEG- DSPE is about 25:70:4: 1.
  • the mixture of lipids includes DOTAP, SPC, and TPGS, for example in a molar ratio of about 25:70:5.
  • the invention is also a method of diagnosing or treating a cancer or infectious disease, by administering an effective amount of a pharmaceutical composition as described herein to a patient in need thereof.
  • the cancer treated can be, for example, brain cancer, bladder cancer, lung cancer, breast cancer, melanoma, skin cancer, epidermal carcinoma, colon and rectal cancer, non-Hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney (renal cell) cancer, prostate
  • cancer leukemia thyroid cancer, head and neck, ovarian cancer, hepatocellular cancer, cervical cancer, sarcomas, gastric cancers, multiple myeloma, lymphomas, and gastrointestinal cancer, and uterine cancer.
  • the cancer is breast cancer, epidermal carcinoma, or pancreatic cancer.
  • Figure 1 illustrates HIF- ⁇ mRNA down-regulation in MDA-MB-435 cells upon treatment with L-RX-0047 and cRGD-L-RX-0047.
  • Figure 2 illustrates HIF- ⁇ mRNA expression in KB cells upon treatment with free RX- 0047, L-RX-0047 and LCAN-RX-0047.
  • Figure 3 illustrates Akt- 1 mRNA down-regulation in KB cells upon treatment with LCAN-
  • FIG. 4 illustrates Akt-1 mRNA down-regulation in Panc-1 cells upon treatment of LCAN- RX-0201.
  • Figure 5 illustrates in vivo tumor inhibition in KB xenograft tumor model.
  • Mice (5 mice per group) were injected intravenously with 3 mg/kg of PBS, free RX-0047, L-RX-0047 or LCAN-RX- 0047 four times every three day (Q3Dx4). Tumor dimensions were determined by measurement with a caliper every 3-4 days.
  • Figure 6 illustrates in vivo HIF- ⁇ mRNA expression in a KB xenograft tumor model.
  • Mice (5 mice per group) were injected intravenously with 3 mg/kg of PBS, free RX-0047, L-RX-0047 or LCAN-RX-0047 four times every three day (Q3Dx4).
  • Intratumoral expression of HIF- ⁇ mRNA was determined by real-time RT-PCR.
  • Figure 7 illustrates animal survival upon treatment with of LCAN-RX-0047 in KB xenograft tumor model.
  • Mice (10 mice per group) were injected intravenously with 3 mg/kg of PBS, free RX- 0047 or LCAN-RX-0047 four times every three day (Q3Dx4).
  • lipid nanoparticles with improved transfection activity.
  • the lipid nanoparticles may partition hydrophobic molecules within the lipid membrane and/or encapsulate water-soluble particles within the aqueous core.
  • the LN formulations can comprise a single lipid or a mixture of lipids, generally including a charged lipid and a neutral lipid, and optionally further including a PEGylating lipid and/or cholesterol.
  • the LN formulations of the present disclosure may include albumin-polymer conjugates.
  • the lipid nanoparticles comprise hyper-cationized albumin-polycation conjugates (APCs).
  • the LNs can have a diameter of less than 300 nm, or typically between about 50 nm and about 200 nm.
  • LNs according to the invention can exhibit one or more advantages such as enhanced transfection and reduced cytotoxicity, especially under high serum conditions found during systemic administration.
  • the LNs are applicable to a wide range of current therapeutic agents and systems, and can exhibit serum stability, targeted delivery, and/or high transfection efficiency.
  • lipid nanoparticle refers to any vesicles formed by one or more lipid components.
  • the LN formulations described herein may include cationic lipids.
  • Cationic lipids are lipids that carry a net positive charge at any physiological pH. The positive charge is used for association with negatively charged therapeutics such as ASOs via electrostatic interaction.
  • Suitable cationic lipids include, but are not limited to: 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Choi); l,2-dioleoyl-3-trimethylammonium-propane (DOTAP); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); dimethyldioctadecylammonium bromide salt (DDAB); l,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride (DL-EPC); N-[l-(2, 3-dioleyloyx) propyl] -N-N-N-trimethyl ammonium chloride (DOTMA); N-[l-(2, 3-dioleyloyx) propyl] -N-N-N-dimethyl ammonium chloride (DODMA);
  • LIPOFECTIN from GIBCO/BRL
  • LIPOFECT AMINE from GIBCO/MRL
  • siPORT NEOFX from Applied Biosystems
  • TRANSFECTAM from Promega
  • TRANSFECTIN from Bio- Rad Laboratories, Inc.
  • Other cationic lipids known in the art or developed subsequently may also be used in the invention. The skilled practitioner will recognize that many more cationic lipids are suitable for inclusion in the inventive LN formulations.
  • the cationic lipids of the present disclosure may be present at concentrations ranging from about 0 to about 60.0 molar percent of the lipids in the formulation, or from about 5.0 to about 50.0 molar percent of the lipids in the formulation.
  • formulation refers to the lipid-coated albumin nanoparticle (LCAN) that includes the lipid nanoparticle and the cationized albumin-polymer conjugates identified herein that contain nucleic acids.
  • the formulation also includes the targeting agent, when present.
  • the LN formulations presently disclosed may include anionic lipids.
  • Anionic lipids are lipids that carry a net negative charge at physiological pH. These lipids, when combined with cationic lipids, are used to reduce the overall surface charge of LNs and introduce pH-dependent disruption of the LN bilayer structure, facilitating nucleotide release by inducing nonlamellar phases at acidic pH or induce fusion with the cellular membrane.
  • anionic lipids include, but are not limited to: fatty acids such as oleic, linoleic, and linolenic acids; cholesteryl hemisuccinate (CHEMS); l,2-di-0-tetradecyl-sn-glycero-3-phospho-(l '-rac-glycerol) (Diether PG); l,2-dimyristoyl-sn-glycero-3-phospho-(l '-rac-glycerol) (sodium salt); 1 ,2-dimyristoyl-sn-glycero- 3-phospho-L-serine (sodium salt); l-hexadecanoyl,2-(9Z,12Z)-octadecadienoyl-sn-glycero-3- phosphate; l,2-dioleoyl-sn-glycero-3-[phospho-rac-(l -glycerol)]
  • anionic lipids known in the art or developed subsequently may also be used in the invention.
  • the anionic lipids of the present disclosure are present at concentrations ranging from about 0 to about 60.0 molar percent of the formulation, or from about 5.0 to about 25.0 molar percent of the formulation.
  • hydrophilic molecules such as polyethylene glycol (PEG) may be conjugated to a lipid anchor and included in the LNs described herein to discourage LN aggregation or interaction with membranes.
  • Hydrophilic polymers may be covalently bonded to lipid components or conjugated using crosslinking agents to functional groups such as amines.
  • Suitable hydrophilic polymers for conjugation and hydrophilic polymer conjugates include, but are not limited to: polyvinyl alcohol (PVA); polysorbate 80; l ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE- PEG2000); D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS);
  • PVA polyvinyl alcohol
  • DSPE- PEG2000 l ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000
  • TPGS D-alpha-tocopheryl polyethylene glycol 1000 succinate
  • DMPE-PEG2000 dimyristoylphosphatidylethanolamine-PEG2000
  • dipalmitoylphosphatidlyethanolamine-PEG2000 dipalmitoylphosphatidlyethanolamine-PEG2000 (DPPE-PEG2000).
  • DPPE-PEG2000 dipalmitoylphosphatidlyethanolamine-PEG2000
  • Other hydrophilic polymers and conjugates known in the art or developed subsequently may also be used in the invention.
  • the hydrophilic polymer may be present at concentrations ranging from about 0 to about 15.0 molar percent of the formulation, or from about 5.0 to about 10.0 molar percent of the formulation.
  • the molecular weight of the hydrophilic polymer used, such as PEG can be from about 100 and about 10,000 Da, from about 100 and about 5,000 Da or from about 100 to about 2,000 Da.
  • the LNs described herein may further comprise neutral and/or amphipathic lipids as helper lipids. These lipids are used to stabilize the formulation, reduce elimination in vivo, or increase transfection efficiency.
  • the LNs may be formulated in a solution of saccharides such as, but not limited to, glucose, sorbitol, sucrose, maltose, trehalose, lactose, cellubiose, raffinose, maltotriose, dextran, or combinations thereof, to promote lyostability and cryostability.
  • Neutral lipids have zero net charge at physiological pH.
  • One or a combination of several neutral lipids may be included in any LN formulation disclosed herein.
  • Suitable neutral lipids include, but are not limited to: phosphatidylcholine (PC), phosphatidylethanolamine, ceramide, cerebrosides, prostaglandins, sphingomyelin, cephalin, cholesterol, diacylglycerols, glycosylated diacylglycerols, prenols, lysosomal PLA2 substrates, N-acylglycines, and combinations thereof.
  • PC phosphatidylcholine
  • ceramide cerebrosides
  • prostaglandins prostaglandins
  • sphingomyelin cephalin
  • cholesterol diacylglycerols
  • glycosylated diacylglycerols prenols
  • lysosomal PLA2 substrates N-acylglycines, and combinations thereof.
  • lipids include, but are not limited to: phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and
  • sterols such as cholesterol, demosterol, sitosterol, zymosterol, diosgenin, lanostenol, stigmasterol, lathosterol, and dehydroepiandrosterone
  • sphingolipids such as sphingosines, ceramides, sphingomyelin, gangliosides, glycosphingolipids, phosphosphingolipids, phytoshingosine; and combinations thereof.
  • the LN formulations described herein may further comprise fusogenic lipids or fusogenic coatings to promote membrane fusion.
  • suitable fusogenic lipids include, but are not limited to, glyceryl mono-oleate, oleic acid, palmitoleic acid, phosphatidic acid, phosphoinositol 4,5-bisphosphate (PIP2), and combinations thereof.
  • PIP2 phosphoinositol 4,5-bisphosphate
  • Other fusogenic lipids known in the art or developed subsequently may also be used in the invention.
  • the LN formulations described here may further comprise cationic polymers or conjugates of cationic polymers.
  • Cationic polymers or conjugates thereof may be used alone or in combination with lipid nanocarriers.
  • Suitable cationic polymers include, but are not limited to: polyethylenimine (PEI); pentaethylenehexamine (PEHA); spermine; spermidine; poly(L-lysine); poly(amido amine) (PAMAM) dendrimers; polypropyleneiminie dendrimers; poly(2-dimethylamino ethyl)- methacrylate (pDMAEMA); chitosan; tris(2-aminoethyl)amine and its methylated derivatives; and combinations thereof.
  • PEI polyethylenimine
  • PEHA pentaethylenehexamine
  • spermine spermine
  • spermidine poly(L-lysine)
  • PAMAM poly(amido amine) dendrimers
  • Cationic polymers or conjugates known in the art or developed subsequently may also be used in the invention.
  • Chain length and branching are considerations for the implementation of polymeric delivery systems.
  • High molecular weight polymers such as PEI having a molecular weight of about 25,000 are used as transfection agents, but suffer from
  • Low molecular weight polymers such as PEI having a molecular weight of about 600, may not cause cytotoxicity, but can be of limited use due to an inability to facilitate stable
  • Conjugation of low molecular weight polymers to larger particles such as albumin is a thus a useful method of increasing activity of nucleic acid condensation while lowing cytotoxicity in formulations.
  • Anionic polymers may be incorporated into the LN formulations presently disclosed as well.
  • Suitable anionic polymers include, but are not limited to: poly(propylacrylic acid) (PPAA);
  • poly(glutamic acid) PGA
  • alginates PGA
  • dextrans xanthans
  • derivatized polymers and combinations thereof.
  • anionic polymers known in the art or developed subsequently may also be used in the invention.
  • the LN formulation includes conjugates of polymers.
  • the conjugates may be crosslinked to targeting agents, lipophilic moieties, peptides, proteins, or other molecules that increase the overall therapeutic efficacy.
  • Suitable crosslinking agents include, but are not limited to: N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP); dimethyl
  • DTBP 3,3'-dithiobispropionimidate
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropyl carbodiimide
  • EDC l-ethyl-3-[3-dimethylaminopropyl]carbodiimide
  • Sulfo- NHS N-hydroxysulfosuccinimide
  • CDI N'-N'-carbonyldiimidazole
  • CDI N-ethyl-5-phenylisoxazolium-3 'sulfonate
  • LN preparation is suitable to synthesize the LNs of the present disclosure, including methods known in the art. For example, ethanol dilution, freeze-thaw, thin film hydration, sonication, extrusion, high pressure homogenization, detergent dialysis,
  • microfluidization tangential flow diafiltration, sterile filtration, and/or lyophilization may be utilized. Additionally, several methods may be employed to decrease the size of the LNs. For example, homogenization may be conducted on any devices suitable for lipid homogenization such as an Avestin Emulsiflex C5. Homogenized LNs may be recycled back into circulation for extended homogenization. Extrusion may be conducted on a Lipex Biomembrane extruder using a
  • polycarbonate membrane of appropriate pore size 0.05 to 0.2 ⁇ . Multiple particle size reduction cycles may be conducted to minimize size variation within the sample and achieve a desired size.
  • the resultant LNs may then be passed through a Sepharose CL4B to remove excess reagents or processed by tangential flow diafiltration.
  • LNs described herein may further include ethanol in the LN suspension.
  • ethanol in the LN suspension.
  • the incorporation of about 10-40% ethanol in LN formulations permeabilizes the lipid bilayer. Disruption of the lipid bilayer aids in condensation with charged moieties such as ASO and siRNA.
  • LNs prepared in this manner are diluted before administration to reduce the effects of cellular membrane lysis due to the presence of ethanol.
  • ethanol may be removed by dialysis and diafiltration, which also removes non-encapsulated nucleic acid.
  • the LNs can be sterilized. This may be achieved, for example, by passing of the LNs through a 0.2 or 0.22 ⁇ sterile filter with or without pre-filtration.
  • LNs Physical characterization of the LNs can be carried through many methods. Dynamic light scattering (DLS) or atomic force microscopy (AFM) can be used to determine the average diameter and its standard deviation. Ideally, LNs should fall under 200 nm in diameter. Zeta potential measurement via zeta potentiometer is useful in determining the relative stability of particles. Both dynamic light scattering analysis and zeta potential analysis may be conducted with diluted samples in deionized water or appropriate buffer solution. Cryogenic transmission electron microscopy (Cryo-TEM) and scanning electron microscopy (SEM) may be used to determine the detailed morphology of LNs.
  • DLS Dynamic light scattering
  • AFM atomic force microscopy
  • Zeta potential measurement via zeta potentiometer is useful in determining the relative stability of particles. Both dynamic light scattering analysis and zeta potential analysis may be conducted with diluted samples in deionized water or appropriate buffer solution.
  • LNs described herein are stable under refrigeration for several months. LNs requiring extended periods of time between synthesis and administration may be lyophilized using standard procedures. A cryoprotectant such as 10% sucrose may be added to the LN suspension prior to freezing to maintain the integrity of the formulation. Freeze drying loaded LN formulations is recommended for long term stability.
  • cationic polymers are useful to nucleic acid delivery systems.
  • the most well-characterized polymeric transfection agent is high molecular weight polyethylenimine, a large polymer with a molecular weight of about 25kDa, referred to herein as PEI25K.
  • PEI25 has had great success in delivering pDNA to cells; however, cytotoxicity has limited its use.
  • Less toxic, low molecular weight PEI having a molecular weight of about 600 kDa has also been investigated, but this has shown diminished ability to condense and deliver nucleic acids.
  • APCs hyper-cationized albumin- polymer conjugates
  • APCs may either be used alone to deliver agents such as pDNA or combined with lipid-based formulations to deliver agents such as siRNA or ASOs.
  • Albumin also possesses endosomal lytic activity due to its hydrophobic core, which upon conformational change can be exposed and can induce bilayer disruption or membrane fusion.
  • Albumin-PEI600 conjugates have an ionization profile that is responsive to pH change. The charge density is increased at endosomal pH.
  • an APC is combined with a cationic lipid combination to assemble a cationic lipid-APC-nucleic acid nanoparticle.
  • an APC is combined with an anionic lipid combination to assemble a lipid-APC-nucleic acid nanoparticle.
  • the lipid nanoparticles comprise hyper-cationized albumin-polycation conjugates. These lipid nanoparticles have high transfection efficiency without additional cytotoxicity.
  • a low molecular weight pentaethylenehexamine is conjugated to human serum albumin via cross linking agents, resulting in a hyper-cationized pH-responsive APC, also referred to herein as HSA-PEHA.
  • the PEHA-to-HSA ratio is between 1 and 30, preferably 5-20, even more preferably 8-15, even more preferably between 10-12.
  • the resulting formulation that includes the lipid nanoparticle and the incorporated hyper-cationized pH-responsive conjugate such as HSA-PEHA is referred to herein as a lipid-coated albumin nanoparticle (LCAN).
  • An exemplary LCAN is a lipid coated albumin nanoparticles which is composed of DOTAP/sPC/TPGS/HS A-PEHA.
  • LCANs are especially useful for the delivery of NAs, such as antisense oligonucleotides, pDNAs, siR As, shRNAs, miRs, and anti-miRs.
  • HSA-PEHA improves the stability and biological activity of the nanoparticles.
  • the lipids in this formulation are DOTAP, SPC, and TPGS.
  • the ratio of DOTAP:SPC:TPGS is about 25:70:5 (m/m).
  • the weight ratio of total lipids-to-HSA-PEHA is between 20 and 1, for example, between 15 and 2, or between 12.5 and 2.5.
  • targeting agents can provide increased efficacy over passive targeting approaches.
  • Targeting involves incorporation of specific targeting moieties such as, but not limited to, ligands or antibodies, cell surface receptors, peptides, lipoproteins, glycoproteins, hormones, vitamins, antibodies, antibody fragments, prodrugs, and conjugates or combinations of these moieties.
  • targeting agents include folate, cRGD (e.g., cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGDfC)) peptides, galactose-containing moieties, transferrin, EPPTl peptide, low density lipoprotein, epidermal growth factors, and antibodies.
  • cRGD can refer to any derivative of or related cRGD peptide, for example, cRGDfC, cRGDfK, cRGDfE, etc.
  • the cRGD peptide is cRGDfC (cyclo(Arg-Gly-Asp-D-Phe-Cys)).
  • maximization of targeting efficiency can be achieved by surface coating the LN with an appropriate targeting moiety rather than encapsulation of the targeting agent. This method can optimize interaction of the LN with cell surface receptors. Targeting agents may be either directly incorporated into the LN during synthesis or added in a subsequent step.
  • Targeting moieties that do not have lipophilic regions cannot readily insert into the lipid bilayer of the LN directly and may require prior conjugation to lipids before insertion or may form an electrostatic complex with the LNs.
  • a targeting ligand may not be capable of directly binding to a lipophilic anchor.
  • a molecular bridge in the form of a crosslinking agent may be utilized to facilitate the interaction.
  • a crosslinking agent can be useful in situations where steric restrictions of the anchored targeting moiety prevent sufficient interaction with the intended physiological target.
  • linking to a lipid anchor via crosslinking agent may be beneficial.
  • Traditional methods of bioconjugation may be used to link targeting agents to LNs.
  • Reducible or hydrolysable linkages may be applied to prevent accumulation of the formulation in vivo and subsequent cytotoxicity.
  • RGD or cRGD or folate targeting agent is incorporated as a targeting conjugate, for example, folate-PEG-CHEMS (folate- polyethylene glycol-cholesteryl hemisuccinate) or folate-PEG-DSPE (folate-polyethylene glycol- distearoyl phosphatidylethanolamine) or cRGDfC-PEG-DSPE (cyclo(RGDfC)-polyethylene glycol- distearoyl phosphatidylethanolamine).
  • a minimum of 5 mole % of the conjugate contains a targeting agent.
  • the conjugate includes at least about 50 mole %, at least about 80 mole %, at least about 90 mole %, or at least about 95 mole % of the targeting agent. In other exemplary embodiments, the conjugate includes about 50 mole %, about 80 mole %, about 90 mole %, or about 95 mole % of the targeting agent. In exemplary embodiments, the mole percent of targeting conjugate among total lipids is about 0.05 to 20 mole %, for example about 0.5 to 5 mole %.
  • therapeutic and diagnostic agents may be used in conjunction with the LNs described herein.
  • Non-limiting examples of such therapeutic and diagnostic agents include nucleic acids, proteins, polysaccharides, lipids, radioactive substances, therapeutic agents, prodrugs, and combinations thereof.
  • Therapeutic agents include, but are not limited to, antineoplastic agents, anti-infective agents, local anesthetics, anti-allergics, antianemics, angiogenesis, inhibitors, beta-adrenergic blockers, calcium channel antagonists, anti-hypertensive agents, anti-depressants, anti-convulsants, anti-bacterial, anti-fungal, anti-viral, anti-rheumatics, anthelminithics, antiparasitic agents, corticosteroids, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-diabetic agents, anti-epileptics, anti-hemmorhagics, anti-hypertonics, antiglaucoma agents, immunomodulatory cytokines, sedatives, chemokines, vitamins, toxins, narcotics, imaging agents, and combinations thereof.
  • Nucleic acid-based therapeutic agents are highly applicable to the LN formulations of the present disclosure.
  • examples of such nucleic acid-based therapeutic agents include, but are not limited to: pDNA, siRNA, miRNA, anti-miRNA, antisense oligonucleotides (ASO), and
  • modifications to the substituent nucleic acids and/or phosphodiester linker can be made. Such modifications include, but are not limited to: backbone modifications (e.g., phosphothioate linkages); 2' modifications (e.g., 2'-0-methyl substituted bases); zwitterionic modifications (6'- aminohexy modified ODNs); the addition of a lipophilic moiety (e.g., fatty acids, cholesterol, or cholesterol derivatives); and combinations thereof.
  • the therapeutic agent is an ASO targeted to a portion of a nucleic acid encoding Akt-1, and which modulates the expression of Akt-1.
  • the oligonucleotide compounds are designed to specifically hybridize with one or more nucleic acids encoding Akt-1.Such ASOs are disclosed in US patent 7,122,527, the contents of which are hereby incorporated by reference in their entirety.
  • RX-0201 (Archexin ® ) which is a 20-mer phosphorothioate antisense oligonucleotide, is targeted to a site in the coding region of the Akt-1 gene having the following sequence: 5' cgccaaggagatcatgcagc 3' at site 1,478 of Akt-1 gene (Seq. Id. No.: 3).
  • the sequence for the backbone of RX-0201 is complementary to this site.
  • Another ASO, RX-0194 is targeted to a site on the Akt-1 gene having the following sequence: 5' agtggactggtgggggctgg 3' at site 1,271 of Akt-1 gene (Seq. Id. No.: 4).
  • the sequence for the backbone of RX-0194 is complementary to this site.
  • Oligomers comprising either 5 or 10 nucleotide upstream and downstream from the sequence where the 20-mer of RX-0194 was derived showed a measurable inhibition of Akt- 1 mRNA expression.
  • the truncated versions of RX-0194 and RX-0201 also showed an inhibition of cancer cell proliferation.
  • five additional antisense oligonucleotide compounds which down-regulate Akt-1 mRNA expression and cause cytotoxic effects on cancer cell lines include:
  • RX-0616 comprising 5' agatagctggtgacagacag 3' (Seq. Id. No.: 5) hybridizable to the site beginning at position 2101 of Akt-1 gene, having the following sequence: 5' ctgtctgtcaccagctatct 3' (Seq. Id. No.: 6);
  • RX-0627 comprising 5' cgtggagagatcatctgagg 3' (Seq. Id. No.: 7) hybridizable to the site beginning at position 2473 of Akt-1 gene, having the following sequence: 5' cctcagatgatctctccacg 3' (Seq. Id. No.: 8);
  • RX-0628 comprising 5' tcgaaaggtcaagtgctac 3' (Seq. Id. No.: 9) hybridizable to the site beginning at position 2493 of Akt-1 gene, having the following sequence: 5' gtagcacttgaccttttcga 3' (Seq. Id. No.: 10); RX-0632, comprising 5' tggtgcagcggcagcggcag 3' (Seq. Id. No.: 1 1) hybridizable to the site beginning at position 2603 of Akt-1 gene, having the following sequence: 5' ctgccgctgccgctgcacca 3' (Seq. Id. No.: 12); and
  • RX-0638 comprising 5' ggcgcgagcgcgggcctagc 3' (Seq. Id. No.: 2) hybridizable to the site beginning at position site 170 of Akt-1 gene, having the following sequence: 5'
  • the therapeutic agent is an ASO targeted to a portion of a nucleic acid encoding HIF-1, and which modulates the expression of HIF-1.
  • the oligonucleotide compounds are designed to specifically hybridize with one or more nucleic acids encoding HIF-1.
  • ASOs are disclosed in US patent 7,205,283, the contents of which are hereby incorporated by reference in their entirety.
  • HIF-1 a is a potent inhibitor of "Hypoxia inducible factor- 1 alpha" (HIF-1 a) and is targeted to a site on the HIF-1 gene having the following sequence: 5' ttggacactggtggctcatt 3' at site 2,772 of HIF-1 gene (Seq. Id. No.: 14).
  • the sequence for the backbone of RX-0047 is complementary to this site.
  • RX-0149 is targeted to a site in the coding region of the HIF-1 gene having the following sequence: 5' gacttggagatgttagctcc 3' at site 1 ,936 of HIF-1 gene (Seq. Id. No.: 16).
  • the sequence for the backbone of RX-0149 is complementary to this site. Oligomers comprising either 5 or 10 nucleotides upstream and downstream from the sequence where the 20-mer of RX-0047 and RX-0149 were derived showed a measurable inhibition of HIF-1 mRNA expression and an inhibition of proliferation of cancer cells.
  • the truncated versions of RX-0047 and RX-0149 which showed some inhibition of HIF-1 mRNA expression also showed an inhibition of cancer cell proliferation.
  • the present invention includes other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics.
  • the antisense compounds can include from about 10 to about 30 nucleobases, for example, oligonucleotides having about 20 nucleobases (i.e. about 20 linked nucleosides).
  • a nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3 Or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • the antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • the lipid nanoparticles disclosed herein may be designed to favor characteristics such as increased interaction with nucleic acids, increased serum stability, lower RES-mediated uptake, targeted delivery, or pH sensitive release within the endosome.
  • any one of the several methods provided herein may be applied to achieve a particular therapeutic aim.
  • Cationic lipids, anionic lipids, PEG-lipids, neutral lipids, fusogenic lipids, cationic polymers, anionic polymers, polymer conjugates, peptides, targeting moieties, and combinations thereof may be applied to meet specific aims.
  • lipid nanoparticles described herein can be used as platforms for therapeutic delivery of oligonucleotide (ON) therapeutics, such as cDNA, siRNA, shRNA, miR A, anti-miR, and antisense oligonucleotides (ASO).
  • ON oligonucleotide
  • This therapeutics could be used to manage a wide variety of diseases such as various types of cancers, leukemias, viral infections, and other diseases.
  • the particular disease treatable according to the invention depends, of course, upon the therapeutic agent incorporated into the LN of the invention.
  • the invention is particularly suitable for encapsulation of nucleic acids, for example antisense oligonucleotides. Nucleic acids, and in particular antisense nucleotides are especially useful for the treatment of tumors and cancers.
  • tumors and cancers treatable according to the invention include, for example Brain cancer, bladder cancer, lung cancer, breast cancer, melanoma, skin cancer, epidermal carcinoma, colon and rectal cancer, non- Hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney (renal cell) cancer, prostate cancer, leukemia thyroid cancer, head and neck, ovarian cancer, hepatocellular cancer, cervical cancer, sarcomas, gastric cancers, multiple myeloma, lymphomas, and gastrointestinal cancer, and uterine cancer .
  • Specific examples include epidermal carcinoma, pancreatic cancer and breast cancer.
  • Targeting moieties such as cRGD peptides, folate, transferrin (Tf), antibodies low density lipoprotein (LDL), and epidermal growth factors can greatly enhance activity by enabling targeted drug delivery.
  • Multi-targeted systems are another possibility and may be applied further to specify a particular target cell subtype.
  • the LNs described herein may be administered by the following methods: peroral, parenteral, intravenous, intramuscular, subcutaneous,
  • the LNs are delivered intravenously, intramuscularly, subcutaneously, or intratumorally. Subsequent dosing with different or similar LNs may occur using alternative routes of administration.
  • compositions of the present disclosure comprise an effective amount of a lipid nanoparticle formulation disclosed herein, and/or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier for pharmaceutically acceptable carriers.
  • compositions that produce no adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human.
  • preparation of a pharmaceutical composition that contains at least one compound or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by
  • compositions disclosed herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • Compositions disclosed herein can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneal ly, intranasally, intravaginally, intrarectal ly, topically, intramuscularly, subcutaneously, mucosally, in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in creams, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference).
  • the actual dosage amount of a composition disclosed herein administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic
  • the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200
  • milligram/kg/body weight about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a derivable range from the numbers listed herein a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
  • a composition herein and/or additional agents is formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the
  • compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier.
  • a composition described herein may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally (U.S. Patents 6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641 ,515; and 5,399,363 are each specifically incorporated herein by reference in their entirety).
  • compositions disclosed herein as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate or gelatin.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person
  • Sterile injectable solutions are prepared by incorporating the compositions in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • compositions may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or via inhalation.
  • topical i.e., transdermal
  • mucosal administration intranasal, vaginal, etc.
  • inhalation via inhalation.
  • compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin.
  • Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture.
  • Transdermal administration of the compositions may also comprise the use of a "patch.”
  • the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.
  • the compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described in U.S. Patents 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in their entirety).
  • the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Patent 5,725, 871, specifically incorporated herein by reference in its entirety) are also well- known in the pharmaceutical arts and could be employed to deliver the compositions described herein.
  • transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Patent 5,780,045 (specifically incorporated herein by reference in its entirety), and could be employed to deliver the compositions described herein.
  • compositions disclosed herein may be delivered via an aerosol.
  • aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
  • Liposomal formulation for RX-0047 was prepared by an ethanol diffusion method.
  • Lipids composition was DOTAP/DOPE/TPGS or DOTAP/soyPC/TPGS at molar ratio of 45:50:5. Briefly, lipids were dissolved in ethanol with or without PEI2K, i.e. PEI having a molecular weight of about 2000. The ratio for lipids-to-PEI2K was 12.5: 1.
  • RX-0047 was dissolved in citrate buffer (20 mM, pH 4) and then added into lipids solution or lipids/PEI2K solution under vortexing to spontaneously form pre-liposomes at an ethanol concentration of 40% (v/v).
  • the weight ratio for RX-0047 to lipids was 12.5: 1.
  • the complexes were then dialyzed against citrate buffer (20 mM, pH 4) at room temperature for 2 h and then against HEPES buffered saline (HBS, 20 mM HEPES, 145 mM NaCl, pH 7.4) overnight at room temperature, using a MWCO 10 000 Dalton Spectra/Por Float- A-Lyzer instrument (Spectrum Laboratories, Collinso Dominguez, CA) to remove free RX-0047.
  • Folate targeted liposomal formulation for RX-0047 was prepared by the same method as described above.
  • the liposomes were composed with DOTAP/sPC/TPGS/F-PEG- CHEMS at molar ratio of 45/50/4.5/0.5 or 45/50/4/1.
  • RGD targeted liposomal formulation for RX-0047 (cRGD-L-RX-0047) was prepared by the same method as described above.
  • the liposomes were composed with DOTAP/sPC/TPGS/cRGD- PEG-DSPE at molar ratio of 45/50/4.5/0.5 or 45/50/4/1
  • HSA-PEHA conjugates were synthesized by activation of carboxyls on HSA with EDC and forming amide linkages with amines on PEHA.
  • the HSA:PEHA:EDC molar ratio used during synthesis was 1 : 1500:200 (mol/mol).
  • HSA (25%, Purchased from Octapharma) was conjugated to pentaethylenehexamine (PEHA, purchased from Sigma- Aldrich) by reacting HSA with a large excess of PEHA in the presence of l-ethyl-3-(3-dimethylamino)-propylcarbodiimide (EDC) and sulfo-N-hydroxysuccinimide in 50 mM borate buffer or water at pH 8.0.
  • EDC pentaethylenehexamine
  • PEHA MW 232.37, technical grade
  • ddH 2 0 ddH 2 0
  • pH 8.0 1 M HC1.
  • EDC l-ethyl-3-[3-dimethylaminopropyl]carbodiimide
  • the HSA-PEHA product was purified by gel membrane chromatography on a PD-10 desalting column or by dialysis using MWCO 10,000 Spectrum membrane against ddH 2 0 (doubly distilled water) at 4 °C to remove unreacted PEHA and byproducts.
  • the dialysis buffer was replaced every 3-4 h until amines from PEHA became undetectable by the standard ninhydrin or trinitrobenzenesulfonic acid (TNBS) amine essay in the external buffer at the 3 h time point at the end of the dialysis cycle.
  • TNBS trinitrobenzenesulfonic acid
  • the dialysis procedure should be replaced by tangential flow diafiltration, e.g., using a Millipore Pellicon cassette system or a Spectropor hollowfiber system.
  • This method can also be used to concentrate the product to a desirable concentration.
  • the product was passed through a 0.22 ⁇ sterile filter into a sterile container and stored at 4 °C.
  • the product can be stored at -20 °C.
  • the product can also be lyophilized.
  • the product protein concentration was determined by BCA protein assay and the PEHA content in the product was determined by TNBS amine content assay using a PEHA-based standard curve.
  • the molecular weight of the HSA-PEHA conjugate was determined by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MADLI TOF MS). On average, there were 1 1 PEHA linked to each HSA based on the result showing m/z of 66405.756. SDS-PAGE analysis showed that the conjugate migrated as a single band, indicating lack of intermolecular crosslinking in the HSA-PEHA product.
  • the cRGDfC and PEG-PSPE- maleimide molar ratio used during the reaction was 1.5: 1.
  • the product was purified by gel filtration on a PD-10 column to remove unreacted/excess cRGDfC from the product.
  • the gel filtration can be replaced with GPC, dialysis using MWCO 2000 membrane, or tangential flow diafiltration.
  • the product can be frozen or lyophilized for long-term stability.
  • the product purity was confirmed by HPLC and by LC-MS.
  • Minimum cRGDfC conjugation level e.g., 80%
  • free peptide content e.g., ⁇ 1 %) can be established as specifications.
  • the cRGDfC content in the product can be determined by BCA protein assay.
  • DSPE-PEG-amine 77 mg was dissolved in DMSO (0.5 ml) with a small amount of triethylamine (20 ⁇ ) and then reacted with folate-NHS synthesized above at molar ratio of 1 :1 for 3hr at room temperature.
  • Folate-PEG-DSPE was purified by precipitation with lOx volume acetone. The precipitate was collected by
  • the product was further purified by dialysis against ddH 2 0 using a MWCO 2000 membrane, followed by lyophilization. Product purity was confirmed by HPLC. Minimum folate conjugation level (e.g., 80%) relative to all PEGylated lipid and undetectable free folate content were established as specifications. The folate content in the product was determined by UV spectrometry at 371 nm or by HPLC.
  • folate-PEG-CHEMS was performed by reacting folate-PEG-amine with CHEMS-NHS. Both folate-PEG-amine and CHEMS-NHS were synthesized by method described previously (Xiang et al, Int J Pharm., 356 (2008) 29-36). Briefly, for synthesis of folate-PEG-bis- amine, folic acid (26.5 mg) and PEG-bis-amine (167.5 mg) were dissolved in 1 mL DMSO. Then, 8.6 mg of NHS and 15.5 mg of DCC were added to the solution, and the reaction was allowed to proceed overnight at room temperature. The product, folate-PEG-amine, was then purified by Sephadex G-25 gel-filtration chromatography.
  • CHEMS-NHS CHEMS (1 g) was reacted with 475 mg NHS and 1.25 g DCC in tetrahydrofuran overnight at room temperature. The product CHEMS-NHS was purified by recrystalization. Finally, for synthesis of F-PEG-CHEMS, folate-PEG-amine (137 mg, 40 ⁇ ) and CHEMS-NHS (29.2 mg, 50 ⁇ ⁇ ⁇ ) were dissolved in CHC1 3 (50 mL), and reacted overnight at room temperature. The solvent (CHC1 3 ) was then removed by rotary evaporation and the residue was hydrated in 50 mM Na 2 C0 3 (10 mL) to form F-PEG- CHEMS micelles.
  • the micelles were then dialyzed against deionized water using a Spectrum dialysis membrane with a molecular weight cut-off (MWCO) of 14 kDa to remove low molecular weight by-products.
  • MWCO molecular weight cut-off
  • the product F-PEG-CHEMS was then dried by lyophilization, which yielded a yellow powder product (130 mg) with a yield of 76.5%.
  • the identity of the product was confirmed by thin-layer chromatography (TLC) and by ⁇ NMR in DMSO-d 6 . 6.
  • Lipid coated albumin nanoparticles were prepared by ethanol dilution method. Lipids l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Avanti Polar Lipids), L-a- phosphatidylcholine derived from soybean (SPC) (Avanti Polar Lipids), and d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) (Eastman Chemical) were dissolved in ethanol. Lipids were combined at 25:70:5 (mol/mol). HSA-PEHA was mixed with lipid solution at weight ratio of 12.5:3. The composition of antisense/total lipid/HSA-PEHA weight ratio was 1 : 10:3.
  • DOTAP Adioleoyl-3-trimethylammonium-propane
  • SPC L-a- phosphatidylcholine derived from soybean
  • TPGS d-alpha-tocopheryl polyethylene glycol 1000 succinate
  • Lipids were combined
  • HSA-PEHA in Hepes buffer (20mM, pH 7.4) and lipids dissolved in EtOH were mixed under vortexing and resulting EtOH concentration was 60%.
  • RX-0047 or RX-0201 was dissolved in Hepes buffer (20mM, pH 7.4) and then added into lipids and HSA-PEHA solution under vortexing to spontaneously form pre-LCAN at an EtOH concentration of 40% (v/v).
  • the complexes were then dialyzed against Hepes buffer (20 mM, pH 7.4) at room temperature for 2 h and then against HEPES buffered saline (HBS, 20 mM HEPES, 145 mM NaCl, pH 7.4) overnight at room
  • the LCAN formulation was concentrated to 20-fold and then washed with Hepes buffer
  • RGD targeted LCAN formulation was prepared by the similar method as described in Example 5. For the RGD targeted LCAN formulation, the lipids were composed with
  • DOTAP/soyPC/TPGS/cRGDfC-PEG-DSPE at molar ratio of 25:70:4: 1 and the composition of antisense/total lipid/HSA-PEHA weight ratio was 1 : 10:3.
  • the lipid mixture containing a targeting agent was made by mixing DOTAP, soyPC, TPGS and cRGDfC-PEG-DSPE as a ratio of 25 :70.4: 1.
  • the lipid mixture components in 60% ethanol (Solution A) were combined with an equal volume of HSA-PEHA in 20% ethanol (Solution B) by two-pumps and a Y-connector to yield a solution of 40% ethanol (Solution C).
  • the RGD targeted LCAN product Solution F was purified by tangential flow diafiltration, MWCO 30 kDa membrane in which included the concentration of the Solution F to 0.5 mg/mL in RX-0201 concentration as a first, diafiltration against 5 mM phosphate buffer (pH 7.4) until the RX-0201 concentration in the permeate solution drops below 10 ⁇ g/mL as a second step and the concentration of the product to 2.5 mg/mL in RX-0201 concentration as a final step. To the product, 1/4 volume of 50% sucrose was added to produce a solution of 10% sucrose and filtered through a 0.22 ⁇ sterile filter; use pre-filtration if necessary.
  • the filtered product (lOmL) was transferred into 50 mL vials, frozen and lyophilized using a 2-stage program: shelf cool to 0 °C, cool 0.5 °C /min to -40 °C, reduce pressure to 0.12-0.16 atm, 30 h primary drying at -25°C. Heat to 25 °C at 5 °C/min, 6h maximum vacuum drying.
  • the final cRGD targeted LCAN product was stored at 4 °C and reconstituted with water for injection at the time of use.
  • LCAN products such as folate-LCAN-RX-0201, cRGD-LCAN-RX-0047 and folate-LCAN-RX-0047) were prepared by the same method as described above.
  • RGD targeted liposomal formulation was composed with DOTAP/sPC/TPGS/cRGDfC- PEG-DSPE at molar ratio of 45/50/4.5/0.5 or 45/50/4/1.
  • the particle size and zeta potential were shown in Table 1.
  • cRGDfC-PEG-DSPE concentration did not change liposome particle size and zeta potentials.
  • Drug loading efficiency in LCAN products was at 2 mg/mL as an oligonucleotide concentration, determined by OliGreen ssDNA quantitation reagent (Invitrogen) as shown in Table 2. The percent recovery of oligonucleotide in the product was 60 to 76%.
  • the particle size of LCAN products was analyzed on a NICOMP Particle Sizer Model 370 (Particle Sizing Systems, Santa Barbara, CA) and ranged 92.7 to 124nm. A volume-weighted Gaussian distribution analysis was used to determine the mean particle diameter and size distribution.
  • the zeta potential ( ⁇ ) was determined on a ZetaPALS (Brookhaven Instruments Corp., Worcestershire, NY). All
  • Example 3 Freeze and thaw stability and lyophilization
  • Example 4 Biological tests 1. mRNA and protein down-regulation by liposomal formulation and LCAN formulation in cancer cells
  • KB human epidermal carcinoma
  • PANC-1 human pancreas
  • MDA-MB-435 human breast cells
  • KB cells were grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, lOOU/ml penicillin and 100 mg/ml streptomycin.
  • PANC-1 and MDA-MB-435 cells were grown in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, lOOU/ml penicillin and 100 mg/ml streptomycin.
  • FBS heat-inactivated fetal bovine serum
  • lOOU penicillin
  • Cells were plated at a density of 2x10 5 cells/well into 6-well plates and cultured overnight.
  • Cells were transfected with L-RX-0047 or.F-L-RX-0047 or cRGDfC-L-RX-0047 for 4 h at 37 °C.
  • 100 uM of folic acid or cRGDfC was added to media during F-L-RX- 0047 or cRGDfC-L-RX-0047 exposure.
  • the medium was replaced with fresh growth medium and the cells were incubated for 48 h at 37 °C under 5% C0 2 atmosphere. Then cells were collected and analyzed for HIF-la mRNA level by real-time qRT-PCR and for HIF-la protein (nuclear protein) expression by western blot analysis.
  • HIF-l mRNA down-regulation by treatment of L-RX-0047 or cRGDfC-L-RX-0047 was determined by real-time RT-PCR (Figure 1) at 0.25 ⁇ of RX-0047 concentration. The results showed that cRGDfC-L-RX-0047 decreased HIF-l mRNA expression in MDA-MB-435 cells. cRGDfC-L-RX-0047 with 1.0% of cRGDfC showed more HIF- 1 a mRNA down-regulation compared to cRGDfC-L-RX-0047 with 0.5% cRGDfC.
  • LCAN-RX-0047 significantly decreased HIF-l mRNA expression compared to liposomal formulation containing RX-0047 (L-RX-0047) at 0.5 ⁇ of RX-0047 concentration ( Figure 2). Also, L-RX-0047 showed significant HIF-l mRNA down-regulation compared to free RX-0047 or control group.
  • RX-0201 was evaluated in KB and Panc-1 cells.
  • LCAN-RX-0201 significantly decreased Akt-1 mRNA expression compared to LCAN-control at 1 ⁇ of RX-0201 concentration ( Figure 3).
  • Panc-1 cells treated by LCAN-RX-0201 showed the significant decrease of Akt-1 mRNA expression compared to LCAN-control or free RX-0201 at 1 ⁇ of RX- 0201 concentration ( Figure 4).
  • LCAN-RX-0047 In vivo gene targeting efficiency of LCAN-RX-0047 was evaluated in KB xenograft tumor model. As shown in Figure 5, free RX-0047 at dose of 3mg/kg only slightly decreased tumor volume relative to the PBS control, although the difference was not significant. L-RX-0047 significantly decreased tumor growth compared to PBS control or free RX-0047 at dose of 3mg/kg. In contrast, LCAN-RX-0047 was more significantly decreased tumor growth than L-RX-0047.
  • ILS (mean survival time of the treated mice/mean survival time of control mice - 1) x l00%.
  • Mice (10 mice per group) were injected intravenously with 3 mg/kg of PBS, free RX-0047 or LCAN-RX-0047 four times every three day (Q3Dx4).
  • the median survival time (MeST) for PBS, free RX-0047, or LCAN-RX-0047 groups was 19, 26, and 37 days, respectively ( Figure 7). Percentage ILS values are 94.7% for LCAN-RX-0047.
  • 2 out of 10 mice were completely cured following treatment with LCAN-RX-0047.

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